PROJECT ABSTRACT: Illnesses caused by the tick-borne rickettsiales of the genera Ehrlichia and Anaplasma have been a growing health concern in recent years. These illnesses include human monocytic ehrlichiosis, human granulocytic ehrlichiosis and human granulocytic anaplasmosis which are caused by Ehrlichia chaffeensis, E. ewingii and Anaplasma phagocytophilum, respectively. Despite the complex cellular environment of arthropods and vertebrates having sophisticated systems of defense, the rickettsiales evolved strategies to evade host clearance. For example, E. chaffeensis in macrophages and tick cells differ significantly in their morphology with distinct patterns of protein expression. The expression patterns include the differential expression from the p28-Omp multigene locus which contains 22 genes. The p28 Omp gene expression is restricted primarily to the p28-Omp gene 19 in macrophages, whereas in tick cells the expressed antigen is predominantly from the p28-Omp gene 14. Our studies also demonstrate that the differential expression in vertebrate and tick cells contributes to the altered host response, such as the delayed clearance in a vertebrate host for tick cellderived E. chaffeensis compared to that for macrophage-derived bacteria. This study will focus on the basic understanding of the regulation of gene expression from the p28-Omp multigene locus. The central hypothesis of this study is that the transcription from the p28-Omp locus in E. chaffeensis is differentially regulated in response to environmental signals and that the host cell-specific gene expression is essential for pathogen[unreadable]s survival in vertebrate and tick cell environments. Specific aims of this study are;1) establish in vitro transcription and translation system for mapping Ehrlichia promoters, 2) map transcriptional machinery in differentially expressed genes 14 and 19 of the p28-Omp locus, 3) evaluate the contributions of macrophage and tick cell environments for differential expression from genes 14 and 19, and 4) evaluate knockout mutations for the p28-Omp genes 14 and 19 to assess the biological relevance of host cell specific expression. The results from this study will provide important information for understanding E. chaffeensis gene regulation and how the rickettsiale in macrophages and tick cells respond to the loss of expression from a differentially expressed protein.